CN111205220B - Fluorescent probe and preparation method and application thereof - Google Patents
Fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN111205220B CN111205220B CN202010121330.1A CN202010121330A CN111205220B CN 111205220 B CN111205220 B CN 111205220B CN 202010121330 A CN202010121330 A CN 202010121330A CN 111205220 B CN111205220 B CN 111205220B
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title abstract description 11
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 claims abstract description 144
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 239000003054 catalyst Substances 0.000 claims abstract description 7
- UKVQBONVSSLJBB-UHFFFAOYSA-N 2-pyridin-2-ylacetonitrile Chemical compound N#CCC1=CC=CC=N1 UKVQBONVSSLJBB-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000010438 heat treatment Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 24
- 239000000523 sample Substances 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 229940126062 Compound A Drugs 0.000 claims description 8
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 7
- 239000001632 sodium acetate Substances 0.000 claims description 7
- 235000017281 sodium acetate Nutrition 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000002689 soil Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
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- 239000012472 biological sample Substances 0.000 claims description 2
- 238000000799 fluorescence microscopy Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 230000004044 response Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- IKHMSRLYUOQHQE-UHFFFAOYSA-N 4-(diethylamino)-2-(2,4-dinitrophenoxy)benzaldehyde Chemical compound CCN(CC)C1=CC=C(C=O)C(OC=2C(=CC(=CC=2)[N+]([O-])=O)[N+]([O-])=O)=C1 IKHMSRLYUOQHQE-UHFFFAOYSA-N 0.000 abstract description 3
- 230000003287 optical effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 5
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- WGAXJEXVOSVLFY-UHFFFAOYSA-N 1-(2,4-dinitrophenoxy)-2,4-dinitrobenzene Chemical group [O-][N+](=O)C1=CC([N+](=O)[O-])=CC=C1OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O WGAXJEXVOSVLFY-UHFFFAOYSA-N 0.000 description 3
- BMVSAKPRNWZCPG-UHFFFAOYSA-N 2-pyridin-4-ylacetonitrile Chemical compound N#CCC1=CC=NC=C1 BMVSAKPRNWZCPG-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 229960000956 coumarin Drugs 0.000 description 3
- 235000001671 coumarin Nutrition 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
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- 108010024636 Glutathione Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 238000005882 aldol condensation reaction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- 206010017758 gastric cancer Diseases 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000011775 sodium fluoride Substances 0.000 description 2
- 235000013024 sodium fluoride Nutrition 0.000 description 2
- 239000004317 sodium nitrate Substances 0.000 description 2
- 235000010344 sodium nitrate Nutrition 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
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- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
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- 206010028372 Muscular weakness Diseases 0.000 description 1
- 208000001738 Nervous System Trauma Diseases 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- -1 aryl sulfide Chemical compound 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- 238000001917 fluorescence detection Methods 0.000 description 1
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- 239000003068 molecular probe Substances 0.000 description 1
- 208000028412 nervous system injury Diseases 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
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- 239000000575 pesticide Substances 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
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- 238000007086 side reaction Methods 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/57—Nitriles
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses aA fluorescent probe and a preparation method thereof, wherein the fluorescent probe has a chemical structural formula in a formula (1):
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a fluorescent probe and a preparation method and application thereof.
Background
Thiophenol (C)6H5SH, PhSH) is an aryl sulfide with high reactivity and has wide applications in the synthesis industry, such as synthesis of sulfonamides, pesticides and macromolecules. On the other hand, thiophenol is highly toxic and respiratory and dermal uptake can lead to a variety of conditions such as dyspnea, muscle weakness, central nervous system injury and even death. The U.S. occupational safety and health administration defines a working environment for thiophenol of 0.1 ppm maximum. With the emission and toxic action of a large amount of industrial use of thiophenol, the development of a method for detecting thiophenol with high sensitivity and high selectivity is very necessary for environmental detection and protection.
The fluorescence method based on the molecular probe has the advantages of simple sample treatment, low cost, simple and quick operation and the like, and is developed and utilized in recent years. However, the existing fluorescent probe molecules for detecting thiophenol have the problems of complex synthesis, insufficient selectivity and sensitivity, poor water solubility and the like (Zhang W.J. Dyes and Pigments 2016, 133, 248; Pagidi S. Langmuir 2018, 34, 8170; Genga Y. Sensors & actors: B. Chemical 2018, 273, 1670.), and therefore, the development of thiophenol detection probes with excellent performance is urgently needed to overcome the problems.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background art, and provide a fluorescent probe which is easy to prepare, stable in performance, excellent in analysis performance and good in water solubility, and a preparation method and application thereof.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a fluorescent probe having the chemical formula in formula (1):
when the fluorescent probe is designed, the water solubility of the fluorescent probe is enhanced by introducing the pyridine group into molecules, and meanwhile, in consideration of the molecular characteristics that thiophenol has stronger nucleophilic reaction capability and can react with electron-deficient molecules or groups, the 2, 4-dinitrophenyl ether group is introduced during molecular design, and the 2, 4-dinitrophenyl ether group is used as a response group of the thiophenol by utilizing the characteristic that the 2, 4-dinitrophenyl ether can react with the thiophenol in a specific manner; when the colorimetric method or the fluorescent method is adopted for detection, thiophenol can attack phenoxy of the fluorescent probe in a nucleophilic mode, and the phenolic hydroxyl group of the probe can be liberated through further removing reaction, and due to the molecular design of the probe, the liberated phenolic hydroxyl group can further perform intramolecular addition and cyclization reactions with cyanogen in the fluorescent probe, so that coumarin fluorescent molecules are obtained; the coumarin fluorescent molecule has the advantages of good stability, high luminous intensity, good water solubility and the like, and the reaction active site and the spectral property can be regulated, which is also the reason for designing the fluorescent probe into the structure of the molecular framework with the coumarin parent structure.
Based on the same technical concept, the invention also provides a preparation method of the fluorescent probe, which comprises the following steps:
(1) preparing raw materials;
(2) dissolving the compound A, pyridine acetonitrile and a catalyst in a solvent, and heating for reaction for 1-24 h to obtain a mixed solution; compound a has the chemical structure in formula (2):
(3) and removing the solvent from the mixed solution, and separating by using a chromatographic column to obtain the fluorescent probe.
The fluorescent probe is obtained by aldol condensation reaction of 4- (diethylamino) -2- (2, 4-dinitrophenoxy) -benzaldehyde and pyridine, and the reaction flow of the specific preparation method is as follows:
according to the technical scheme, the compound A, namely 4- (diethylamino) -2- (2, 4-dinitrophenoxy) -benzaldehyde and pyridine acetonitrile are selected as raw materials, the two raw materials are wide in source and low in cost, the yield of the fluorescent probe obtained through reaction is high, the reaction condition is mild, equipment is simple, the steps are simple and convenient, theoretical guidance is provided for industrial production of the fluorescent probe, and the fluorescent probe prepared by the method has the advantages of being stable in performance, excellent in analysis performance and good in water solubility.
Preferably, the reaction time in step (2) is 5 hours.
Preferably, the catalyst is one or more of piperidine, acetic acid, sodium acetate and triethylamine; further, the catalyst is piperidine. The catalyst has a good catalytic effect on aldol condensation reaction, and can improve the reaction efficiency and the final yield of the fluorescent probe.
Preferably, in the step (2), the reaction temperature is 50-120 ℃; further, the reaction temperature in step (2) was 80 ℃. If the reaction temperature is too low, the reaction may be incomplete, and if the temperature is too high, side reactions are likely to occur, which affects the yield of the final product.
Preferably, the solvent is one or more of acetonitrile, ethanol, sodium acetate and triethylamine.
Based on the same technical concept, the invention also provides an application of the fluorescent probe, and the fluorescent probe is applied to the determination of thiophenol in water bodies, soil and biological systems.
Preferably, the fluorescent probe is applied to the detection of thiophenol in water or soil, and specifically comprises the following steps:
(1) drawing a standard curve: dissolving the fluorescent probe in a phosphoric acid mixed buffer system to obtain a probe solution; adding thiophenol with different concentrations into the probe solution, measuring the fluorescence intensity of the probe solution after adding the thiophenol under a specific wavelength, and drawing a standard curve according to the obtained fluorescence emission intensity to the concentration of the thiophenol in the probe solution;
(2) preparing a water body or soil sample to be detected into a solution to be detected, adding a fluorescent probe into the solution to be detected, measuring the fluorescence intensity of the solution to be detected under a specific wavelength, and obtaining the concentration of thiophenol in the solution to be detected according to the fluorescence intensity and a standard curve.
As the optimization of the technical scheme, the fluorescent probe is applied to the determination of thiophenol in a biological system, and specifically comprises the following steps: and (3) culturing the sample of the biological system and the fluorescent probe together, and analyzing the cell biological sample by using a fluorescence imaging method.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a fluorescent probe and a preparation method thereof, and also provides application of the fluorescent probe in the field of thiophenol detection;
(2) the fluorescent probe molecule of the invention has good stability (the fluorescent probe mother solution can be stored for more than three months in a room, the spectral property of the fluorescent probe mother solution is kept unchanged) and optical performance, the single fluorescent probe solution has no fluorescence emission before reaction, and the fluorescent probe has no fluorescence emission along with the addition of thiophenolThe needle molecules have strong fluorescence emission properties at the position of 510 nm; the fluorescent probe has high selectivity and high sensitivity, strong recognition capability on the thiophenol, high response speed and response range of 0-10 mu mol.L-1Low detection limit (16 nmol. L)-1) When the kit is used for detecting thiophenol, various performances are excellent;
(3) the preparation method of the fluorescent probe provided by the invention has the advantages of easily available raw materials, high synthesis yield (up to more than 85%), mild reaction conditions, simple equipment and simple steps, and provides theoretical guidance for industrial production of the fluorescent probe.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent probe in example 1;
FIG. 2 is a graph showing the fluorescence emission intensity of the fluorescent probe solution after adding thiophenol in example 4 versus the concentration of thiophenol;
FIG. 3 is a graph showing a standard curve of the concentration of p-thiophenol in the fluorescence intensity of example 4;
FIG. 4 shows the results of the measurement of the selectivity of the fluorescent probe for thiophenol in example 4;
FIG. 5 is an image of the fluorescent probe used in example 5 to detect thiophenol in human gastric cancer cells (MGC-803).
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Synthesis of fluorescent probe:
example 1:
compound A (0.76 g, 2.12 mmol) and 4-pyridineacetonitrile (0.25 g, 2.12 mmol) were dissolved in absolute ethanol (10 mL), piperidine (0.1mL) was added to the reaction mixture, and the reaction was refluxed at 80 ℃ for 5 hours. After the reaction was completed, the filtrate was removed by suction filtration, and the cake was retained and purified by column chromatography using dichloromethane/ethyl acetate (1:1, volume ratio) as eluent, followed by rotary drying under reduced pressure to obtain 0.838g of a yellow solid (yield 86%). After the product is analyzed by nuclear magnetic resonance hydrogen spectrum, the structural formula is shown as the formula (1):
wherein the structural formula of the compound A is shown as the formula (2):
the hydrogen spectrum of nuclear magnetic resonance is shown in figure 1, and the description of the spectrum is as follows:
1H NMR (400 Hz, CDCl3): 1H NMR (400 MHz, CDCl3): δ 8.96(s, 1H)，8.71 (s, 2H)，8.54(s, 1H)，8.11 (s, 1H)，7.51(s, 2H)，7.44 (s, 1H)，7.08 (s, 1H)，6.90(s, 1H)，6.51 (s, 1H)，2.50 (q, 4H)，1.13 (t, 6H)。MS [ESI]: m/z, calcd for [M+H]+460.15; found 459.58。
example 2:
compound A (0.76 g, 2.12 mmol) and 4-pyridineacetonitrile (0.25 g, 2.12 mmol) were dissolved in anhydrous acetonitrile (10 mL), and triethylamine (0.1mL) was added to the reaction mixture to carry out a reaction at 50 ℃ for 5 hours. After the reaction was completed, the filtrate was removed by suction filtration, and the cake was retained and purified by column chromatography using dichloromethane/ethyl acetate (1:1, volume ratio) as eluent, followed by rotary drying under reduced pressure to obtain 0.563g (yield: 57%) of a yellow solid.
Example 3:
compound A (0.76 g, 2.12 mmol) and 4-pyridineacetonitrile (0.25 g, 2.12 mmol) were dissolved in absolute ethanol (10 mL), and sodium acetate (0.1g) was added to the reaction solution, followed by reflux reaction at 80 ℃ for 12 hours. After the reaction was completed, the filtrate was removed by suction filtration, and the cake was retained and purified by column chromatography using dichloromethane/ethyl acetate (1:1, volume ratio) as eluent, followed by rotary drying under reduced pressure to obtain 0.746g (yield 76%) of a yellow solid.
Fluorescence detection of a fluorescent probe to thiophenol:
example 4:
the fluorescent probe prepared in example 1 was dissolved in a mixed buffer solution of phosphoric acid (10 mM, pH 7.4) to prepare 10. mu. mol. L-1The probe solution of (1). To a 3 mL cuvette, 2mL of 10. mu. mol. L prepared-1Then adding thiophenol with different concentrations, mixing uniformly, and testing the fluorescence spectrum, the result is shown in figure 2. The concentration of thiophenol was plotted by the fluorescence emission intensity at 510nm of the solution, and the obtained standard curve is shown in FIG. 3, and the thiophenol concentrations were 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. mu. mol. L from a to k, respectively-1The solution system is a phosphoric acid mixed buffer solution (10 mM, pH 7.4) of water, the abscissa is the wavelength, and the ordinate is the fluorescence intensity; as can be seen from the figure, the concentration of thiophenol is 0 to 10. mu. mol. L-1When the concentration is within the range, the fluorescence emission intensity and the thiophenol concentration present a good linear relation, and the quantitative detection of the thiophenol within the concentration range can be realized.
Mu. mol. L of 100. mu. mol of the fluorescent probe prepared in example 1 was added to 10. mu.M-1After various substances (thiophenol, cysteine, glutathione, homocysteine, sodium carbonate, aniline, phenol, sodium acetate, sodium fluoride and sodium nitrate), the fluorescence emission intensity of the mixed solution was measured at 510nm and fluorescence was measured with the test interfering substance as abscissaPlotting the intensity as a vertical coordinate to obtain a test result graph of the selectivity of the fluorescent probe to the thiophenol, which is shown in figure 4; as can be seen, the fluorescent probe prepared in example 1 is not affected by other common substances, such as cysteine, glutathione, homocysteine, sodium carbonate, aniline, phenol, sodium acetate, sodium fluoride and sodium nitrate. Under the condition of the existence of the interference ions, the probe still has good selectivity and sensitivity to the phenol containing the thiophenol.
Detection of thiophenol in cells by fluorescent probes:
example 5:
cultured human gastric cancer cells (MGC-803) were incubated at 37 ℃ in 10. mu.M PBS solution of the fluorescent probe prepared in example 1 for 1 hour to obtain a cell image, which was free of fluorescence. The fluorescent image of MGC-803 cells obtained by culturing the cells in the culture medium containing the fluorescent probe prepared in example 1 at 37 ℃ for 1 hour, adding thiophenol, culturing in a thiophenol-containing solution for 1 hour, washing with PBS buffer solution, and then obtaining the cells is shown in FIG. 5: FIGS. 5 (A) and (B) are a bright field photograph and a fluorescent photograph of MGC-803 cells cultured with the fluorescent probe (10. mu. mol. L-1) prepared in example 1, respectively; (C) and (D) are respectively a bright field picture and a fluorescence picture of MGC-803 cells cultured by the molecular fluorescent probe (10 mu mol. L-1) and thiophenol (20 mu mol. L-1); therefore, the fluorescent probe has good cell membrane permeability and can be used for cell imaging analysis in the biomedical field.
Claims (9)
1. A fluorescent probe having a chemical structure of formula (1):
2. the method for preparing the fluorescent probe according to claim 1, which comprises the following steps:
(1) preparing raw materials;
(2) dissolving the compound A, pyridine acetonitrile and a catalyst in a solvent, and heating for reaction for 1-24 h to obtain a mixed solution; the compound A has a chemical structural formula in formula (2):
(3) and removing the solvent from the mixed solution, and separating by using a chromatographic column to obtain the fluorescent probe.
3. The method of claim 2, wherein the catalyst is one or more of piperidine, acetic acid, sodium acetate, and triethylamine.
4. The method of preparing a fluorescent probe according to claim 2, wherein the solvent of the mixed solution in the step (3) is removed by spin-drying the mixed solution under reduced pressure.
5. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature in the step (2) is 50 to 120 ℃.
6. The method of any one of claims 2 to 5, wherein the solvent is one or more of acetonitrile, ethanol, sodium acetate, and triethylamine.
7. Use of the fluorescent probe of claim 1 for the determination of thiophenol in water, soil and biological systems.
8. The use of the fluorescent probe as claimed in claim 7, wherein the fluorescent probe is used for the determination of thiophenol in water or soil, and comprises the following steps:
(1) drawing a standard curve: dissolving the fluorescent probe in a phosphoric acid mixed buffer system to obtain a probe solution; adding thiophenol with different concentrations into the probe solution, measuring the fluorescence intensity of the probe solution after adding the thiophenol under a specific wavelength, and drawing a standard curve according to the obtained fluorescence emission intensity to the concentration of the thiophenol in the probe solution;
(2) preparing a water body or soil sample to be detected into a solution to be detected, adding the fluorescent probe into the solution to be detected, measuring the fluorescence intensity of the fluorescent probe under the specific wavelength, and obtaining the concentration of thiophenol in the solution to be detected according to the fluorescence intensity and the standard curve.
9. The use of the fluorescent probe of claim 7 for the determination of thiophenol in biological systems, comprising the steps of: and (3) culturing the sample of the biological system and the fluorescent probe together, and analyzing the cell biological sample by using a fluorescence imaging method.
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